Enzymatic esterification process

ABSTRACT

Aliphatic, saturated or unsaturated, straight or branched chain C2-C24 monocarboxylic acids and polyhydric alcohols, particularly glycerol are selectively reached to monoesters in the presence of an enzyme which comprises potato lipid acyl hydrolase. The process can be used too for upgrading technical monoglycerides which contain free fatty acids.

This application is the national phase of international applicationPCT/EP97/02959 filed May 30, 1997 which designated the U.S.

The present invention relates to a process for the preparation ofmono-esters of aliphatic, saturated or unsaturated, straight or branchedchain monocarboxylic acids having from 2 to 24 carbon atoms andpolyhydric alcohols in the presence of an acyl hydrolase.

When a carboxylic acid and a polyol are contacted for an esterificationprocess, a mixture is formed comprising fully esterified polyol andpolyols partially esterified to various degrees: monoesters, diestersetc. It has appeared very difficult to obtain selectively mono-esterwhen various hydroxyl groups on the polyol are available for reaction.

BACKGROUND ART

There have been many investigations in the past for developing aconvenient method for the selective preparation of mono-esters ofpolyols. Both enzymatically catalysed as well as non-enzymaticallycatalysed methods have been investigated. A review of the various waysin which this subject was investigated using enzymatically catalysedprocesses is given by U. T. Bornscheuer in “Enzyme and MicrobialTechnology”, 17, 578-586, 1995.

An example of such a process, using enzymes having esterase activity,such as lipases or esterases, is given in European Patent SpecificationEP-B-0,215,038 (Novo Industri A/S). In this patent a process for thepreparation of monoglycerides has been described in which first twohydroxyl groups of glycerol are blocked by converting them into a ketalor an acetal, such as isopropylidene glycerol or glycerol diethylketal.This ketal or acetal is then reacted with a carboxylic acid or acarboxylic acid ester in the presence of an esterase. The acetal orketal protecting group is then removed by acid catalysis from theresulting ester to produce monoglyceride. This synthesis constitutes arather cumbersome route, however, in that first two adjacent hydroxylgroups of the glycerol molecule need to be blocked involving chemicalreaction and the blocking group has then to be removed in a finaldeprotection step.

Therefore, there is still a need for a simple enzymatic process forpreparing fatty acid mono-esters of polyhydric alcohols which iseconomically attractive with regard to the price of the enzyme and whichleads to high yields of monoglyceride and the smallest possible amountof di-esters or higher esters.

A group of closely related glycoproteins, known as patatin, isresponsible for lipid acyl hydrolase activity found in potato tubers.The lipid acyl hydrolase is only known for its activity to catalyze thedeacylation (hydrolysis) of a range of naturally occurring lipids, e.g.monoglycerides, diglycerides and phospholipids (Biochem. J. 121 (3),379-390 (1971)).

The use of a lipid acyl hydrolase for the formation of wax esters fromlong chain monocarboxylic acids and long chain monohydric alcohols hasbeen demonstrated (S. Dennis and T. Galliard, Phytochemistry 13 [11],2469-2473 [1974]). It is surprising that the synthesis of polyolmono-esters, such as monoglycerides, has never been suggested orproposed.

SUMMARY OF THE INVENTION

We have found that a lipid acyl hydrolase occurring inter alia in potatotubers, is particularly suitable for the enzymatic production ofmono-esters of aliphatic carboxylic acids and polyhydric alcohols. Thisenzyme is available in good quantities, because it can relatively easilybe obtained from abundantly available raw materials. Although the tuberscontain the highest amount of said enzyme, lesser amounts can also befound in other parts of the potato plant. The enzyme can also beobtained by applying genetic engineering techniques.

Therefore the present invention relates to a process for the preparationof mono-esters of aliphatic, saturated or unsaturated, straight orbranched chain C2-C24 monocarboxylic acids and polyhydric alcohols inthe presence of an enzyme, wherein the enzyme is potato lipid acylhydrolase, which means that the hydrolase is obtainable from potatoes oris identical in substrate specificity. Said enzyme selectivity catalyzesthe formation of mono-glycerides. Higher esters are formed too, but invery small quantities only.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the increase of acylglycerol content when the enzymaticesterification of carboxylic acid and polyol proceeds. MG ismonoglyceride, DG is diglyceride. Temperature is 40° C., water contentis 3.3 wt. %.

FIG. 2 shows the increase of acylglycerol content when the enzymaticesterification of carboxylic acid and polyol proceeds, with and withoutthe use of vacuum. MG is monoglyceride, DG is diglyceride.

DETAILS OF THE INVENTION

Preferably aliphatic saturated straight chain monocarboxylic acidshaving from 6 to 22 carbon atoms are used.

The polyhydric alcohol is selected preferably from the group consistingof dihydric alcohols, such as the glycols, e.g. ethylene glycol,propylene glycol, dipropylene glycol, trihydric alcohols, such asglycerol, tetrahydric alcohols such as diglycerol, pentahydric alcohols,hexahydric alcohols such as the sugar alcohols and further sugars, sugaralkyl ethers, such as the alkyl glycosides, and mixtures thereof. Theuse of glycerol, diglycerol and C₁-C₁₈ alkylglycosides, such as ethylglycoside, is preferred.

The lipid acyl hydrolase according to the invention may be used in theform of a protein extract isolated from potato tubers, which extract maybe enriched. A specific acyl hydrolase extracted from potatoes is knownas patatin. The protein extract may also be obtained from the potatoleaves. The protein may also be obtained using genetic engineeringtechniques. The genes encoding patatin have been cloned successfully.Hence, the fermentative production of patatin in high yield, using agenetically modified yeast or mould, is also possible.

The total water content of the reaction mixture preferably is kept below10 wt. %, preferably at 0.01-5 wt. %. According to a preferred optionthe water formed during the reaction is removed. This can be achievedusing any technique known in the art, such as pervaporation and vacuumevaporation.

The reaction temperature is between 10° C. and 90° C., preferablybetween 25° C. and 55° C.

The process of the present invention is also suitable for the upgradingof technical grade monoglycerides which contain free fatty acids.Therefore another embodiment of the present invention is a process fordeacidification of a crude monoglyceride comprising reacting themonoglyceride in the presence of glycerol and a catalytic amount ofpotato lipid acyl hydrolase under similar conditions as described above.

The enzyme according to the invention may be used effectively in animmobilized form, e.g. supported on diatomaceous earth particles.

The invention is illustrated with the following examples:

EXAMPLE 1 Preparation of Potato Protein Extract

Potato tubers (variety-Sante) (1.8 kg) were washed, peeled and chippedinto wedges which were immersed immediately into 0.01 wt. % sodiummetabisulphite solution containing 10 wt. % polyvinylpolypyrrolidone(PVPP) (ex Sigma Chemical Co.) After draining off the aqueous mixtureand PVPP, the potato wedges were put into plastic bags and frozen atminus 18° C.

An aliquot of the potato wedges (1.6 kg) was then allowed to thaw andhomogenised in a Waring blender at 4° C. for 1 minute with 1.5 l of 100mM sodium phosphate buffer (pH 7.0) containing 0.02 wt. % sodiummetabisulphite and 1 wt. % PVPP. The homogenate was filtered throughthree layers of muslin, and the filtrate was centrifuged at 8,000 g for30 minutes. The supernatant was dialysed overnight at 4° C. against 10 lof deionised water. The resulting dialysed mixture was centrifuged at18,000 g for 1 hour and the supernatant was shell frozen and reduced toa volume of about 100 ml using a freeze drier. After thawing theconcentrated solution was centrifuged at 18,000 g for 1 hour, and thesupernatant shell frozen and freeze dried to give a powder (7.2 g)containing about 50 wt. % protein.

EXAMPLE 2 Monoglyceride Synthesis from Oleic Acid

Mixtures containing oleic acid (90%, ex Aldrich Chemical Co.) (1.41 g=5mmoles), glycerol (Pricerine™ 9098, ex Unichema International) (0.56g=6.1 mmoles), potato protein extract (100 mg) and various amounts ofwater (0-70 μl) were stirred in stoppered test tubes at varioustemperatures (40-60° C.). Samples were taken from the reaction mixturesperiodically for analysis by GC and TLC.

A typical progress curve for a reaction run at 40° C. is shown in FIG.1. The major products of the reactions were monoglycerides (MG) withonly low levels of diglycerides (DG). Examination of the final productsby TLC showed that no triglycerides were formed.

Table 1 shows the compositions of the products formed after 72 hoursreaction at various temperatures and various amounts of water. It alsoshows the initial reaction rates as calculated from the progress curves.Reaction rates are expressed as micromoles product per minute and pergram of extract. At 40° C. addition of water had little effect on thefinal product yield, but it caused a slight stimulation in the initialreaction rate. Increasing the reaction temperature raised the initialreaction rate. With 3.3 wt. % of water maximum conversion was observedat 50° C. At 60° C. the initial reaction rate was higher, but the finalconversion was lower, which suggests that enzyme inactivation occurredduring reaction at this higher temperature. At 50° C. with 3.3 wt. % ofwater 75% of the oleic acid was converted into an acylglycerols mixtureconsisting of 96 mole % MG and 4 mole % DG.

In the absence of potato extract the rate of acylglycerol formation wasvery slow (<0.01 μmole.min⁻¹ per gram of extract at 60° C.).

EXAMPLE 3 Synthesis of Monoolein Using Vacuum to Improve the Yield

A mixture of oleic acid (1.5 g=5.32 mmoles), glycerol (0.57 g=6.2mmoles), water (70 μl) and potato protein extract (100 mg) was stirredat 50° C. and a vacuum (<50 mbar) was applied to the system using an oilpump to remove water from the mixture. For comparison a similar reactionwas run without vacuum in a stoppered tube. Samples were taken from thereaction mixtures periodically for analysis by GC. Progress curves forthe reactions are given in FIG. 2. In the reaction cariied out undervacuum 4.43 mmoles of MG and 0.10 mmoles of DG were formed after 72hours. 87% of the oleic acid was converted into acylglycerols. In thecomparison reaction after 72 hours 3.51 mmoles of MG and 0.14 mmoles ofDG were formed. The final conversion of oleic acid into acylglycerolswas 71%.

EXAMPLE 4 Synthesis of Monoglycerides from Various Fatty Acids

Mixtures of fatty acid (5.1 mmoles), glycerol (6.1 mmoles), water (70μl) and potato protein extract (50 mg) were stirred at varioustemperatures in stoppered tubes. The products formed after 6 hours wereanalyzed by GC. Table 2 shows the yields of mono- and diglycerides for avariety of saturated and unsaturated fatty acids. For long chainsaturated fatty acids a high reaction temperature (70° C.) was necessaryto melt the reactants. A lower yield of MG was obtained probably becauseof inactivation of the protein catalyst at the high reactiontemperature.

EXAMPLE 5 Preparation of Immobilized Enzyme

Potato protein extract (500 mg) was dissolved in 1.5 ml of phosphatebuffer (pH 7.0, 10 mM). Acid washed, flux calcined diatomaceous earth(1.0 g) (Celite™ ex Manville Corporation) was added to the proteinsolution. After mixing a thick paste was obtained. This paste was driedovernight at room temperature in a vacuum oven to give an immobilizedenzyme powder.

EXAMPLE 6 Synthesis of Monoglycerides Using Immobilized Enzyme Powder

A mixture of oleic acid (1.42 g=5.04 mmoles), glycerol (0.57 g=6.2mmoles), water (70 μl) and immobilized enzyme powder (100 mg) wasstirred in a stoppered tube at 40° C. for 48 hours. Analysis of thereaction product by GC showed the formation of 3.31 mmoles of monooleinand 0.24 mmoles of diolein. The immobilized enzyme powder has appearedto be an effective catalyst for MG synthesis.

TABLE I Effect of temperature and amount of added water on monoglycerideand diglyceride synthesis Initial reaction Products formed rate after 72hour Reaction Amount of water μmoles reaction Temperature added min⁻¹ gMG DG (° C.) (μl) (wt. %) extract⁻¹ (mmoles) (mmoles) 40 0 0 36.4 2.340.09 25 1.2 39.5 2.28 0.16 50 2.4 44.2 2.42 0.21 70 3.3 42.5 2.34 0.2150 70 3.3 64.8 3.51 0.14 60 70 3.3 110.5 2.93 0.09 70 70 3.3 — 0.48 nd

EXAMPLE 7 Synthesis of Diol Monoesters of Oleic Acid

Mixture of oleic acid (7.05 g=25 mmoles), diol (ex Aldrich Chemical Co.)(25 mmoles), water (350 μl) and potato protein extract (500 mg) werestirred at 30° C. in stoppered tubes for 6 hours. The resulting reactionmixtures were analysed by gc. The results given in Table III show thatmonoesters were the major reaction products and only low level ofdiesters were formed.

TABLE II Monoglyceride and diglyceride synthesis from various fattyacids Reaction Products formed after 6 Fatty acid temperatures hoursreaction reactant (° C.) MG (mmoles) DG (mmoles) capric (10:0) 50 1.400.02 lauric (12:0) 50 1.65 0.05 myristic (14:0) 60 3.13 0.04 palmitic(16:0) 70 0.91 0.02 stearic (18:0) 70 0.17 nd oleic (18:1) 50 1.50 0.04linoleic (18:2) 50 1.59 0.03 linolenic (18:3) 50 2.53 0.03

TABLE II Monoglyceride and diglyceride synthesis from various fattyacids Reaction Products formed after 6 Fatty acid temperatures hoursreaction reactant (° C.) MG (mmoles) DG (mmoles) capric (10:0) 50 1.400.02 lauric (12:0) 50 1.65 0.05 myristic (14:0) 60 3.13 0.04 palmitic(16:0) 70 0.91 0.02 stearic (18:0) 70 0.17 nd oleic (18:1) 50 1.50 0.04linoleic (18:2) 50 1.59 0.03 linolenic (18:3) 50 2.53 0.03

EXAMPLE 8 Synthesis of Diglycerol Esters of Oleic Acid

A mixture of oleic acid (1.45 g=5.14 mmoles), diglycerol (ex UnichemaInternational: contains 92% diglycerols, 4% glycerol, 4% other polyols)(0.9 g), water (70 μl) and potato protein extract (100 mg) was stirredat 50° C. and a vacuum (<50 mbar) was applied to the system using an oilpump. After 48 hours the reaction was stopped and the products analysedby GC. The major reaction products were diglycerol monoesters (2.60mmoles). Smaller amounts of diglycerol diesters (0.56 mmoles) and MG(0.18 mmoles) were also formed.

EXAMPLE 9 Synthesis of Propanediol Monoesters of Capric Acid

Mixtures of capric acid (ex Fluka) (0.85 g=5 mmoles), and eitherpropan-1,2-diol or propan-1,3-diol (ex Aldrich Chemical Co.) (0.38 g=5mmoles), water (70 μl) and potato protein extract (100 mg) were stirredat 35° C. in stoppered tubes for 24 hours. The reaction products wereanalysed by GC. With propan-1,2-diol 0.28 mmoles of capric acidmonoesters were formed, while with propan-1,3-diol 1.02 mmoles ofmonoester were produced. Only low levels of diesters were detected inthe reaction products.

EXAMPLE 10 Synthesis of Diglycerol Esters of Capric Acid

A mixture of capric acid (0.86 g=5 mmoles), diglycerol (0.82 g), water(70 μl) and potato protein extract (100 mg) was stirred at 35° C. in astoppered tube for 8 hours. Analysis of the resulting mixture by GCshowed that the major reaction products were capric acid monoesters ofdiglycerol (1.72 mmoles). Smaller amounts of diglycerol diesters (0.24mmoles) and monoglycerides (0.11 mmoles) were also formed.

EXAMPLE 11 Synthesis of Ethyl Glucoside Monocaprate

A mixture of capric acid (0.86 g=5 mmoles), ethylglucoside (ex UnichemaInternational: containing ˜80% ethylglucosides, 7% glucose and 10%diglucosides) (1.04 g), water (70 μl) and potato protein extract (100mg) was stirred at 35° C. in a stoppered tube for 72 hours. Analysis ofthe resulting reaction mixture by GC showed that 0.21 mmoles ofethylglucoside monocaprate was produced. Only trace amounts ofethylglucoside diesters were detected.

EXAMPLE 12 Synthesis of Sorbitol Esters

A mixture of capric acid (0.86 g=5 mmole), sorbitol (ex Aldrich ChemicalCo.) (0.91 g=5 mmole), t-butanol (500 μl), water (70 μl) and potatoprotein extract (100 mg) was stirred at 35° C. in a stoppered tube for72 hours. Analysis of the resulting reaction mixture showed thatsorbitol monocaprate (0.12 mmoles) and sorbitol dicaprate (0.04 mmoles)were formed.

What is claimed is:
 1. A process for deacidification of a monoglyceridecontaining free C2-C24 monocarboxylic acid as impurity which comprisescontacting said monoglyceride with glycerol and a catalytic amount ofpatatin at a temperature of from 25° C. to 50° C. whereby said free acidis removed by esterification of the acid with said glycerol.